ABSTRACTRegulation of MHC class I gene expression is critical to achieve proper immune surveillance. In this work, we identify elements downstream of the MHC class I promoter that are necessary for appropriate in vivo regulation: a novel barrier element that protects the MHC class I gene from silencing and elements within the first two introns that contribute to tissue specific transcription. The barrier element is located in intergenic sequences 3' to the polyA addition site. It is necessary for stable expression in vivo, but has no effect in transient transfection assays. Accordingly, in both transgenic mice and stably transfected cell lines, truncation of the barrier resulted in transcriptional gene silencing, increased nucleosomal density and decreased histone H3K9/K14 acetylation and H3K4 di-methylation across the gene. Significantly, distinct sequences within the barrier element govern anti-silencing and chromatin modifications. Thus, this novel barrier element functions to maintain transcriptionally permissive chromatin organization and prevent transcriptional silencing of the MHC class I gene, ensuring it is poised to respond to immune signaling.

pone-0006748-g008: Histone modifying enzymes and histone variant H2A.Z are associated with PD1 3′ intergenic sequences.(A) ChIP analysis was performed on L cells stably transfected with PD1cDNAint1-2/Bam, as described in Materials and Methods, to determine the extent of association of CTCF, CPB, PCAF, USF1 and USF2 across the PD1 gene, as indicated. All three 3′ UTR primers gave the same results; only 3′UTR(1) is shown. (B) The binding of histone variant H2A.Z across the PD1 gene was similarly determined in the same clone. All results shown are representative of two ChIP experiments of independent clones. None of the 3′UTR primers showed any amplification following ChIP with anti-H2A.Z.

Mentions:
Histone modifying enzymes have been reported to be associated with barrier elements, thereby actively regulating chromatin modifications [31]. In light of the differences in chromatin organization and modifications among the constructs, we assayed by ChIP for the presence of the histone acetyl transferase enzymes, CBP and PCAF across the PD1cDNAint1-2/Bam gene in L cells. Indeed, we found that both CBP and PCAF were associated with the 3′ intergenic region as well as with the promoter (Figure 8A).

pone-0006748-g008: Histone modifying enzymes and histone variant H2A.Z are associated with PD1 3′ intergenic sequences.(A) ChIP analysis was performed on L cells stably transfected with PD1cDNAint1-2/Bam, as described in Materials and Methods, to determine the extent of association of CTCF, CPB, PCAF, USF1 and USF2 across the PD1 gene, as indicated. All three 3′ UTR primers gave the same results; only 3′UTR(1) is shown. (B) The binding of histone variant H2A.Z across the PD1 gene was similarly determined in the same clone. All results shown are representative of two ChIP experiments of independent clones. None of the 3′UTR primers showed any amplification following ChIP with anti-H2A.Z.

Mentions:
Histone modifying enzymes have been reported to be associated with barrier elements, thereby actively regulating chromatin modifications [31]. In light of the differences in chromatin organization and modifications among the constructs, we assayed by ChIP for the presence of the histone acetyl transferase enzymes, CBP and PCAF across the PD1cDNAint1-2/Bam gene in L cells. Indeed, we found that both CBP and PCAF were associated with the 3′ intergenic region as well as with the promoter (Figure 8A).

Bottom Line:
Accordingly, in both transgenic mice and stably transfected cell lines, truncation of the barrier resulted in transcriptional gene silencing, increased nucleosomal density and decreased histone H3K9/K14 acetylation and H3K4 di-methylation across the gene.Significantly, distinct sequences within the barrier element govern anti-silencing and chromatin modifications.Thus, this novel barrier element functions to maintain transcriptionally permissive chromatin organization and prevent transcriptional silencing of the MHC class I gene, ensuring it is poised to respond to immune signaling.

ABSTRACTRegulation of MHC class I gene expression is critical to achieve proper immune surveillance. In this work, we identify elements downstream of the MHC class I promoter that are necessary for appropriate in vivo regulation: a novel barrier element that protects the MHC class I gene from silencing and elements within the first two introns that contribute to tissue specific transcription. The barrier element is located in intergenic sequences 3' to the polyA addition site. It is necessary for stable expression in vivo, but has no effect in transient transfection assays. Accordingly, in both transgenic mice and stably transfected cell lines, truncation of the barrier resulted in transcriptional gene silencing, increased nucleosomal density and decreased histone H3K9/K14 acetylation and H3K4 di-methylation across the gene. Significantly, distinct sequences within the barrier element govern anti-silencing and chromatin modifications. Thus, this novel barrier element functions to maintain transcriptionally permissive chromatin organization and prevent transcriptional silencing of the MHC class I gene, ensuring it is poised to respond to immune signaling.